Typically, whole biological samples that are to be visualized using a microscope are sliced into a thin layer and mounted in order to see the lateral structure. Thus, the sample takes on a two dimensional property by substantially reducing the thickness of the sample, so that the structure within the plane of the slice can be seen without added background from other structures that reside outside of the plane. However, some structures, including many in-vivo structures, can not be sliced.
It is known in the prior art to use confocal fluorescence microscopy to image an in-vivo structure at sub-cellar resolutions. Similarly, it is well known that standard wide-field fluorescence microscopy does not provide optical sectioning for laterally uniform objects. Wide-field techniques are hampered by their inability to reject out-of-focus background structures, thereby leading to low signal contrast.
Techniques have been developed that enable whole-mount and in-vivo samples to be examined by optical sectioning. These techniques all function to minimize or eliminate out-of-focus background (i.e. background arising from structures that are not within the focal plane). Several strategies have been devised to provide for optical sectioning. For example, wide-field microscopy may employ dynamic speckle illumination to obtain an optically sectioned image. When dynamic speckled illumination is used, a fluorescent structure is illuminated with random speckle pattern(s). Optical sectioning is then obtained by calculating the contrast of the image fluctuations in time. Thus, many images must be taken. One problem with this technique is that it is slow and generally requires several tens of images to produce a final sectioned image of reasonable quality.
Other techniques include using a grid pattern to illuminate the structure to be visualized. The pattern illumination is shifted by a portion of the grid period and an image is captured with each shift. The data from the multiple images is processed to remove the out-of-focus background. One drawback of this technique is that the resultant images often have a striped appearance due to motion related artifacts. Since the images are obtained sequentially, any movement of the structure to be visualized or the imaging device results in misregistration between images at a processor and therefore, a degradation of the processed image quality.